Intracellular ionized free calcium, [Ca.sup.2+ ].sub.i, is well recognized in medical and biological research as an important regulator of normal cell function, and may be an important factor in heart disease, cancer, stroke, neuromuscular disease, and other pathologic conditions. As such, it has been the subject of intense investigation within all fields of cellular physiology. To date, however, it has remained difficult to measure [Ca.sup.2+ ].sub.i in living cells.
Four general methods of [Ca.sup.2+ ].sub.i measurement have been used and are currently in use: bioluminescent indicators (eg. aequorin); metallochromic indicators (eg. arsenazo III); fluorescent indicators (eg. quin2); and Ca.sup.2+ -selective microelectrodes. None of these methods have proved completely satisfactory. Ca.sup.2+ -selective microelectrodes have a frequency response too slow to be of use in the measurement of rapid calcium transients, and require impalement of cells which may induce membrane ion leaks. Measurement of [Ca.sup.2+ ].sub.i with the metallochromic dyes is impeded by difficulty in loading cells, by motion artifacts, by interaction with Mg.sup.2+ and H.sup.+, and by difficulties in calibration. The bioluminescent indicator aequorin has proven more satisfactory than other systems of measurement, but is difficult to calibrate. The Ca.sup.2+ -sensitive fluorescent indicator quin2 may be easily loaded into cells in the acetoxymethyl ester form; however, the free dye released into the cell by intracellular esterase action buffers [Ca.sup.2+ ].sub.i transients at dye loadings required for sufficient fluorescent intensity. Current systems are limited to fluorescence measurement only and do not provide for real time visualization of the cell or tissue preparation while fluorescence measurement is taking place.
In view of the unsatisfactory results obtained in current methods of [Ca.sup.2+ ].sub.i measurement, new fluorescent Ca.sup.2+ indicators have recently been identified as having properties more conducive to such measurement. A discussion of those improved dyes used in conjunction with the present invention follows.